PROJECT SUMMARY/ABSTRACT Our goal is to understand molecular principles of germ cell regulation. We focus on a decision that all stem cells make: to self-renew or begin differentiation. This decision is fundamental in all tissues and is critical to human health. Regulators that promote the stem cell state must be active to drive self-renewal and totipotency, but must be inactivated for differentiation; conversely, regulators that are critical for differentiation must be inactive in stem cells, but then become activated at the right time and place for differentiation. Regulation of the transition ? from self-renewal to differentiation ? balances the two states and is fundamental to development, homeostasis and human health. Defects in that balance cause human disease, including tissue atrophy and cancer. RNA regulation is a major conserved theme in germ cells from worms to mammals. Key RNA-binding proteins regulate self-renewal and differentiation across animal phylogeny. We focus on PUF (Pumilio and FBF) RNA- binding proteins, which have pivotal roles in germ cells and neurobiology across animal species. In humans, defective PUF proteins cause infertility and epilepsy. PUF proteins have many features in common: They sculpt the transcriptome and proteome by binding to more than 1000 mRNAs, with 100's conserved between nematodes and humans; they regulate target mRNAs by diverse mechanisms, including activation and repression; and they work in a combinatorial fashion with partner proteins, most of which are also conserved. The challenge now is to understand how PUF proteins regulate the balance between self-renewal and differentiation in molecular terms and in a native in vivo context. Our proposal addresses this challenge with a sharp focus on four issues. First, we elucidate how distinct PUF mechanisms ? activation and repression ? work together to control germ cell fates. Second, we elucidate how PUF partnerships operate in vivo, to understand their logic and glean general principles. Third, we elucidate regulation of the balance between self-renewal to differentiation. Fourth, we elucidate how a functional module of multiple PUF proteins and partners maintains stem cells under diverse physiological and environmental conditions. These four issues are fundamental to all PUF regulation. For each, we take a multidisciplinary and multiscale approach and exploit an exceptionally tractable network. Together, our findings will advance our understanding germ cell regulation with potential for broad impact, including of new therapy designs for humans.